1. Field of the Invention
The present invention relates to a sample plate for laser desorption ionization mass spectrometry and a laser desorption ionization mass spectrometer using the sample plate.
2. Description of Related Art
There has been increased the necessity for a measuring method of molecular weights of high molecular compounds composed of an organism-related substance such as protein, peptide, sugar, oligonucleotide, etc., and synthetic high molecular compounds, and importance is attached to the use of mass spectrometry as the method. In mass spectrometry, a sample is ionized to achieve separation of ions on the basis of a mass-to-charge ratio. In mass spectrometry of high molecular compounds, there are adopted LDI-MS (Laser Desorption Ionization-Mass Spectrometry) and a measuring apparatus therefor.
When a laser beam is irradiated directly on a sample, which is composed of a high molecular compound, in order to ionize the sample, decomposition of the sample is caused. Hereupon, there is generally used a method, in which decomposition of a sample is avoided by coating the sample on a medium, which absorbs a laser beam, or supplying the sample in a mixed state. The ionization method free of decomposition of a sample is called a soft LDI-MS, a typical example of which adopts Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). A mixed solution composed of a low molecular organic compound, which is called a matrix reagent and absorbs a laser beam, and a sample solution is applied on a mass spectrometry sample plate to be dry-crystallized, thus forming a sample on the plate.
In measurement with a mass spectrometer, the sample formed on the plate is mounted in an ion source and a laser beam is irradiated on a surface of the sample. A matrix reagent having absorbed the laser beam efficiently is momentarily vaporized and ionized. At that time, sample molecules taken in as a mixed crystal are vaporized substantially simultaneously and give and receive an electric charge from the matrix reagent whereby the sample molecules are ionized without little decomposition. After ions as generated are mass-separated owing to an action of a mass separation part of a time-of-flight type, a quadrupole type, an ion-trap type, a sector type, a Fourier transformation type, or a composite type thereof, the ions are detected by a detector and mass is analyzed.
Among these, a method of using a mass separation part of a time-of-flight type involves no limit for mass range of measurement in principle, so that Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOFMS) is generally used in mass spectrometry for high molecules.
It is said that when laser desorption ionization is combined with a time-of-flight type mass spectrometer, detection up to immunoglobulin M (molecular weight of ca. 900 kDa) can be made and a limit of detection reaches an amol level. Also, compounds capable of ionization cover a wide range of general organism-related substances such as peptide, protein, polysaccharides, compound lipid, nucleic acid-related substances, etc., synthetic polymer, oligomer, metal coordination compounds, and inorganic compounds. In case of using a matrix, various substances are used as the matrix (“Bunseki”, No. 4, 253 to 261 (1996)).
In order to give a high resolving power, there has been proposed a sample feeding apparatus for analysis of a sample comprising a plate having a flat surface, which gives electroconductivity to the apparatus, and in which the flat surface is electroconductive to have a resistance of less than about 1500 ohm per 1 square inch, the flat surface is coated with a graphite paint, and the plate is made of a material selected from polypropylene, polyethylene, polystyrene, polycarbonate, etc. (JP-A-2003-43014).
There is known a probe, a sample of which placed surface is decorated by means of energy absorptive molecules and which serves as means for enabling mounting and dismounting to bring about favorable results of molecules being an object of analysis without addition of foreign matrix molecules, which is carried out in the related art, the surface containing synthetic polymer, glass, or ceramic (JP-A-2000-131285). Also, there is known a metallic sample plate placed surface having a larger hydrophilic property than that of the metal itself, more specifically, the surface being subjected to hydrophilic treatment such as plasma treatment, etc. (JP-A-2004-347524).
In MALDI-MS described above, a low molecular organic compound is used as an ionizing reagent, so that disturbance ions are generated attributable thereto. Besides, since the disturbance ions thus generated not only cover in the vicinity of a molecular weight (a mass number of 500 or less) of the organic compound but also form a cluster to cover a range of mass number of several thousands or more, analysis becomes difficult in many cases.
Also, since kinds of matrix reagents suited to ionization are different according to samples in MALDI-MS described above, there is an inconvenience that an appropriate matrix reagent must be selected trial and error when samples being measured are prepared.
Therefore, there has been proposed a soft LDI-MS, in which a fine powder inorganic compound is used as an ionizing agent for ionization.
Known as the fine powder inorganic compound are, for example, cobalt fine powder (JP-A-62-43562, JP-A-63-318061), titanium oxide fine powder (C. T. Chen, Y. C. Chen: Anal. Chem., 76, 1453 (2004)), graphite powder (J. Sunner, E. Dratz, Y. C. Chen, Anal. Chem., 67, 4335 (1955)), carbon nanotube (S. Xu, Y. Li, H. Zou. J, Qiu. Z, Guo, B. Guo,: Anal. Chem., 75, 6191 (2003)), a carbon black solid having an average particle size of 100 nm or less and a PVC blackness of 50 or less (JP-A-2000-180413), a support plate for crystallization of an additive for mass spectrometry, made of a layer, at least a surface of which contains carbon, the additive for mass spectrometry being used in combination with α-cyano-4-hydroxy cinnamic acid (JP-A-2001-13110), and the like.
In such a method, in which fine powder is used, a solution-like sample and a suspension solution composed of fine powder are coated on a surface of a sample plate for mass spectrometry, so that it is difficult to uniformly coat a sample and frequently becomes difficult in the case where mass spectrometry is performed with high efficiency. Further, irradiation of a laser beam sometimes disperses an ionization medium in an ion source and contamination thereby causes a problem.
Hereupon, there has been proposed a soft LDI-MS, in which a porous silicon plate is used as a sample plate (J. Wei, J. M. Buriak, G. Siuzdak: Nature, 399, 243 (1999)). This method is called DIOS-MS (desorption/ionization-mass spectrometry on porous silicon). In this method, mass spectrometry is performed by coating a sample solution on a surface of a porous silicon plate having fine holes of nanometer level, mounting the plate in an ion source of a mass spectrometer after drying, and irradiating a laser beam on a sample surface in subsequent operations like MALDI-MS. While a detailed principle of ionization in DIOS-MS is not apparent, it is thought that a nanosilicon structure absorbs a laser beam with high efficiency to be rapidly heated whereby instantaneous desorption of sample molecules occurs, and a component bonded to or absorbed by a porous silicon is ionized to deliver an electric charge to the sample molecules whereby ionization of the sample is attained. Also, there is used a silicon nanowire grown on gold minute particles, which are precipitated on a silicon plate (E. P. Go, J. V. Apon, G. Luo, A. Saghatelian, R. H. Daniels, V. Sahi, R. Dubrow, B. F. Cravatt, A. Vertes, G. Siuzdak: Anal. Chem., 77, 1641 (2005)). Also, there are proposed a plate, a surface portion of which is provided with recesses and made of a plastic material, covered by a metallic film, and subjected to silicon etching, and a chip that uses a sponge-like substance (JP-A-2004-184137).
Since DIOS-MS uses a sample plate itself as an ionization medium, there is produced an advantage that it is relatively easy to uniformly coat a sample, and it is possible to avoid generation of a disturbance peak, which causes a problem in MALDI-MS. Since the efficiency of ionization of a porous silicon is greatly affected by conditions of fabrication and it is extremely difficult to fabricate sample plates having the same porous structure with good reproductivity, it is hard under present circumstances to say that DIOS-MS is a reliable technology of mass spectrometry. Further, since most of samples once coated are taken into a porous structure, a major part of sample molecules is not ionized and remains to make an obstacle in measurement with high sensitivity and cleaning of a sample plate after measurement is not easy and responsible for prevention of generation of a peak attributable to a sample for a previous measurement, so that DIOS-MS is not so much suited to repeated measurement.
Also, in the method of using a silicon nanowire, since gold minute particles making a base material of the nanowire are bonded to a silicon substrate automatically in an unstable state, a nanowire gold minute particle structure is susceptible to failure in irradiation of a laser beam during measurement, or in a cleaning step of a sample plate after measurement, so that DIOS-MS is not so much suited to repeated measurement.
Because of the above, there have been earnestly desired to develop a plate, on which a sample solution can be uniformly coated, and which does not generate any disturbance peak even when a laser beam is irradiated after being coated with the sample solution, is easy to clean after measurement, can be applied to analysis of various kinds of samples, and enables measurement with high sensitivity, a method of laser desorption ionization mass spectrometry, in which a plate coated with the sample solution is used to prevent generation of any disturbance peak, and an apparatus therefor.
It is desirable to provide a sample plate for laser desorption ionization mass spectrometry, which serves for a soft LDI-MS measurement, does not generate any disturbance peak even when a laser beam is irradiated thereon, enables correct mass spectrometry of high sensitivity on various samples such as organism-related substances, synthetic organic compounds, etc. and enables uniformly coating a sample thereon in fabrication of a sample, and of which cleaning after measurement is easy, and a measuring apparatus using the sample plate.
The inventors of the present application have earnestly carried on studies to provide such a sample plate, on which a sample solution can be uniformly coated, and which does not generate any disturbance peak even when a laser beam is irradiated after being coated with the sample solution, and is easy to clean after measurement. They proposed an ionization plate, for which a pyroelectric single crystal plate is used, as a plate coated with the sample solution (Japanese Patent Application No. 2005-013433). but satisfactory results are not produced.